FIGURE 5.
GASP-1 knockdown reduces D2 and D3 receptor degradation. Knockdown of GASP-1 by shRNA directed against GASP-1 (shGASP-1a and shGASP-1b) or a scrambled shRNA (shScr) in D2- (A) and D3 (B)-expressing cells. HEK293 cells stably expressing either D2 (C) or D3 (D) plus shScr (upper panel), shGASP-1a (middle panel), or shGASP-1b (lower panel) were surface-biotinylated and incubated in the absence or presence of either DA (10 μm, D2) or PMA (100 nm, D3) for 30, 90, or 180 min. The fate of the surface-labeled endocytosed receptors was assessed after immunoprecipitation of receptor using anti-FLAG (D2) or anti-HA (D3) antibodies followed by SDS-PAGE and streptavidin overlay. 100% lane shows total surface receptor labeled. Strip lane shows efficiency of thio cleavage. A representative immunoblot is shown. NT, no treatment.